Within the scope of the present invention, a microcarrier or a microparticle refers to any type of particles, respectively to any type of carriers, microscopic in size, typically with the largest dimension being from 100 nm to 300 micrometers, preferably from 1 μm to 200 μm.
According to the present invention, the term microcarrier refers to a microparticle functionalized, or designed to be functionalized, that is containing, or designed to contain, one or more ligands or functional units bound to the surface of the microcarriers or impregnated in its bulk. A large spectrum of chemical and biological molecules may be attached as ligands to a microcarrier. A microcarrier can have multiple functions and/or ligands. As used herein, the term functional unit is meant to define any species that modifies, attaches to, appends from, coats or is covalently or non-covalently bound to the surface of said microcarrier or impregnated in its bulk. These functions include all functions that are routinely used in high-throughput screening technology and diagnostics.
The term microchannel or microfluidic channel refers to a closed channel, i.e. an elongated passage for fluids, with a cross-section microscopic in size, i.e. with the smallest dimension of the cross-section being typically from about 1 to about 500 micrometers, preferably about 10 to about 300 micrometers. A microfluidic channel has a longitudinal direction, that is not necessarily a straight line, and that corresponds to the direction in which fluids are flowing within the microfluidic channel, i.e. preferably essentially to the direction corresponding to the average speed vector of the fluid, assuming a laminar flow regime.
Drug discovery or screening and DNA sequencing commonly involve performing assays on very large numbers of compounds or molecules. These assays typically include, for instance, screening chemical libraries for compounds of interest or particular target molecules, or testing for chemical and biological interactions of interest between molecules. Those assays often require carrying out thousands of individual chemical and/or biological reactions.
Numerous practical problems arise from the handling of such a large number of individual reactions. The most significant problem is probably the necessity to label and track each individual reaction.
One conventional method of tracking the identity of the reactions is achieved by physically separating each reaction in a microtiter plate (microarray). The use of microtiter plate, however, carries several disadvantages like, in particular, a physical limitation to the size of microtiter plate used, and thus to the number of different reactions that may be carried out on the plate.
In light of the limitations in the use of microarray, they are nowadays advantageously replaced by functionalized encoded microparticles to perform chemical and/or biological assays. Each functionalized encoded microparticle is provided with a code that uniquely identifies the particular ligand(s) bound to its surface. The use of such functionalized encoded microparticles allows for random processing, which means that thousands of uniquely functionalized encoded microparticles may all be mixed and subjected to an assay simultaneously. Examples of functionalized encoded microparticles are described in the international patent application WO 00/63695.
The applicant proposed in its international patent application WO 2010/072011 an assay device having at least one microchannel with an inlet and an outlet, the microchannel serving as a reaction chamber in which a plurality of functionalized encoded microparticles or microcarriers 10 (FIG. 1) can be packed. The microfluidic channel is provided with restricting or stopping means acting as a filter that allows a liquid solution containing chemical and/or biological reagents to flow through while blocking the microcarriers 10 inside the microchannel. The microparticles 10 are designed such that their shape and size relative to the cross-section of the microchannel prevent any overlapping of adjacent microcarriers 10. Thus, the microcarriers 10 exhibit a monolayer arrangement inside each microchannel and are stacked onto the restricting means along the microchannel.
Those functionalized encoded microcarriers 10 that show a favorable reaction of interest between their attached ligand(s) and the chemical and/or biological reagents flowing through may then have their code optically read, thereby leading to the identification of the ligand producing the favorable reaction.
The code may comprise a distinctive pattern of a plurality of traversing holes 12 and may also include an asymmetric orientation mark such as, for example, a L-shaped sign 14 (as shown in FIG. 1) or a triangle. This asymmetric orientation mark allows the distinction between the top surface 16 and the bottom surface 18 of the microcarrier 1.
With the assay device described in WO 2010/072011, microparticles are introduced within the microchannel from the inlet and immobilized onto the restricting means. Then, a biological sample (comprising one or more target molecules) is flown in the microchannel (comprising one or more sets of microcarriers) around the microparticles and then through the restricting means toward the outlet, the microcarriers being still blocked by the restricting means. The detection of a reaction of interest can be based on continuous readout of the fluorescence intensity of each encoded microcarrier present in a microfluidic channel. In other words, the presence of a target molecule in the assay will trigger a predetermined fluorescent signal.
However, after the microparticles 10 have been inserted within the microchannel and are stacked onto the restricting means, the microparticles 10 may be slightly offset relative to one another in the direction perpendicular to the microchannel 20, i.e. in the Z-direction (FIG. 2).
Then, when performing the continuous read-out of fluorescence as described in WO2010/072011A1, it has then been observed that some or all of the microparticles 22 may exhibit a non-homogenous intensity over their surface emitting fluorescence light in response to binding of target molecules (FIG. 3).
FIG. 4 is an enlarged view of a microparticle 22 presenting a non-homogenous intensity over its surface. The microparticle 22 clearly exhibits one region 24 having a grey level inferior to a second surrounding region 26.
This non-homogenous intensity is due to non-homogeneous mass transfer in the microchannel which is mainly the consequence of the particular arrangement of microcarriers 10 in the Z-direction within the microchannel 20. Non-homogeneous mass transfer within the microchannel 20 leads to a non-uniform flow of the target molecules on the surface of the microparticles.
The non-homogeneous mass transfer is problematic as it affects the attribution of the fluorescence value to the microcarrier. In case non-homogeneity is significant, the value of fluorescence that is attributed to the microcarrier will not reflect the correct concentration of target molecules within the analyzed sample.
Thus, non-homogeneous mass transfer affects the reliability of the measured signal. Incorrect values on a plurality of microcarriers can lead to serious consequences on the reliability of the assay and therefore on its usefulness in the field of diagnostics, genomic research and molecular biology.